Tracheophytes Contain Conserved Orthologs of a Basic Helix-Loop-Helix Transcription Factor That Modulate <i>ROOT HAIR SPECIFIC</i> Genes

Abstract

<i>ROOT HAIR SPECIFIC</i> (<i>RHS</i>) genes, which contain the root hair-specific <i>cis</i>-element (RHE) in their regulatory regions, function in root hair morphogenesis. Here, we demonstrate that an <i>Arabidopsis thaliana</i> basic helix-loop-helix transcription factor, ROOT HAIR DEFECTVE SIX-LIKE4 (RSL4), directly binds to the RHE in vitro and in vivo, upregulates <i>RHS</i> genes, and stimulates root hair formation in Arabidopsis. Orthologs of RSL4 from a eudicot (poplar [<i>Populus trichocarpa</i>]), a monocot (rice [<i>Oryza sativa</i>]), and a lycophyte (<i>Selaginella moellendorffii</i>) each restored root hair growth in the Arabidopsis <i>rsl4</i> mutant. In addition, the rice and <i>S. moellendorffii</i> RSL4 orthologs bound to the RHE in in vitro and in vivo assays. The <i>RSL4</i> orthologous genes contain RHEs in their promoter regions, and RSL4 was able to bind to its own RHEs in vivo and amplify its own expression. This process likely provides a positive feedback loop for sustainable root hair growth. When <i>RSL4</i> and its orthologs were expressed in cells in non-root-hair positions, they induced ectopic root hair growth, indicating that these genes are sufficient to specify root hair formation. Our results suggest that RSL4 mediates root hair formation by regulating <i>RHS</i> genes and that this mechanism is conserved throughout the tracheophyte (vascular plant) lineage.